This proposal is aimed at understanding the mechanisms and signaling pathways by which down- regulation of lipoprotein lipase (LPL) leads to improved insulin sensitivity in muscle cells. In previous studies, it was demonstrated that chronic treatment of L6 rat myotubes with Thiazolidinediones (TZDs) drastically reduced their expression of LPL with a co-incident increase in glucose uptake. Thus, TZDs may increase insulin sensitivity in muscle, at least partly, by repressing LPL expression and activity. Consistent with this hypothesis, a direct down-regulation of LPL message and protein levels using LPL-specific siRNA resulted in a concurrent increase in insulin-dependent glucose uptake. The effect of LPL silencing on other insulin-regulated metabolic processes will be examined. It will be of great pharmaceutical value if decreasing muscle LPL expression or activity can improve the overall metabolic response of muscle cells to insulin. Short hairpin RNA (shRNA) plasmids will be used to generate a stable LPL-knock-out L6 cell line and study insulin-regulated metabolic pathways including the oxidation and synthesis of fatty acids, and the synthesis of glycogen. In addition to its lipolytic function, LPL is also a ligand for lipoprotein receptors, This is an additional mechanism by which LPL contributes to cellular lipid uptake. It will be determined if the insulin-sensitizing effect requires the down-regulation of LPL's lipolytic function, its binding function, or both. This will be accomplished by using a specific lipolysis inhibitor, tetrahydrolipostatin, or by abolishing cell surface binding of LPL using heparinase treatment. Additionally, insulin sensitivity will be measured after the over- expression of various mutant forms of LPL that either lack catalytic activity (mutants LPLC and S132T), or substrate binding activity (mutant W390A/W393A/W394A). LPL is not a traditional signaling molecule. Thus, it is interesting that its down-regulation is co- incident with increased glucose uptake. PCR arrays are excellent tools to simultaneously examine the expression of multiple genes involved in a signaling pathway. Preliminary PCR array data indicate that siRNA mediated silencing of LPL results in a change in the expression pattern of various genes involved in insulin signaling including Glut4, PI3Kinase, acetyl coA carboxylase, etc. The expression levels of these and other relevant genes involved in insulin-regulated metabolic pathways will be compared in LPL-knock- out and basal L6 cells. This study will identify signaling intermediates that link LPL expression to insulin sensitivity and map a signaling pathway. This experiments proposed here will help with the development LPL-targeted approaches for the management of insulin resistance and type II diabetes.